The development of new and more effective therapeutic agents often requires clinical studies in which the amount of the agent circulating in blood can be monitored. In particular, many anti-cancer agents must maintain a threshold concentration in blood to effectively block the growth and metastasis of their target cancers.
A variety of methods exist for monitoring the levels of drugs in blood. Most common is a method involving high performance liquid chromatography (HPLC). To utilize HPLC, the solute of interest must first be separated from the majority of blood components. This is typically done using a liquid phase extraction process which is often tedious, time consuming, and messy. The multistep liquid phase extraction often involves partitioning the sample between two solvents (usually an aqueous/organic mixture), drawing off and discarding the aqueous solvent, filtering the organic solvent to remove any particulate matter, evaporating the organic solvent and finally reconstituting the residue in a solvent suitable for the subsequent chromatographic separation. The conditions to be used for chromatography involve consideration of numerous parameters. Most important are the stationary phase or column packing and the elution mobile phase.
The materials most commonly used for packings in HPLC columns are microparticulate silicas. These small porous silica particles have either a spherical or irregular shape and a nominal diameter of 3, 5 or 10 .mu.m. Additionally, the silica is often chemically modified or "bonded" with a variety of organic residues to alter the size, shape and pore size of the silica particle, the carbon content of the bonded phase and the extent of end-capping. End-capping is a method used to reduce the residual adsorptive properties of the silica. The result of these modifications is a wide variety of column packings from many manufacturers including a trend toward "application-specific" column packings.
The composition of the mobile phase controls a separation more than any other factor. Most separations utilize a binary solvent mixture as mobile phase, however ternary or quaternary compositions also work. If the composition of the mobile phase is constant, the elution is called "isocratic". Alternatively, the composition of the mobile phase can be made to change in a predetermined manner during the separation by using programmed solvent mixing systems. Elution done in this manner is known as "gradient" elution. Gradient elution is used in situations when the range of column retention times for a series of solutes is so large that they cannot be eluted in a reasonable time using a single solvent or solvent mixture. For example, if a sample contains compounds having a wide range of polarities, the separation can be carried out by changing the polarity of the solvent mixture during separation.
The terms normal phase and reverse phase chromatography are used to describe a number of separations. In normal phase chromatography the polarity of the stationary phase is higher than that of the mobile phase. These parameters are achieved when non-bonded silica is used in adsorption chromatography. Reverse phase means that the polarity of the stationary phase is lower than that of the mobile phase. This result is achieved when the silica is hydrocarbon-bonded and the mobile phase is polar. For either type of chromatography the solutes elute in order of their polarity, the most polar elute first with reverse phase and the least polar elute first with normal phase.
Reverse phase HPLC offers the further advantage that numerous bonded stationary phases are available to increase the scope of separations which can be obtained. Furthermore, the mobile phase is often an aqueous mixture which reduces solvent costs. Methods have been described for the reverse phase HPLC determination of taxol (Longnecker, et al., Cancer Treat. Rep., 71:53-59 (1987)), clobazam (Streete, et al., Ther. Drug Monit., 13:339-344 (1991)), indomethacin (Johnson, et al., Ther. Drug Monit., 14:61-65 (1992)), rufloxacin (Carlucci, et al., Ther. Drug Monit., 13:448-451 (1991)) and phenylpropanolamine (Stockley, et al., Ther. Drug Monit., 13:332-338 (1991)).
A new anti-cancer agent 1, (CAI) 5-amino-1-(4-(4-chlorobenzoyl)-3,5-dichlorobenzyl)-1,2,3-triazole-4-carboa mide, has recently been discovered and is now in clinical trial. This compound has been shown to inhibit signal transduction pathways and cancer growth in human xenograft models in the concentration range 1-10 .mu.g/mL. Quantitation of 1 in blood has been conducted by precipitating plasma samples with trichloroacetic acid, extracting the samples with methylene chloride, reconstituting the concentrated extract and chromatographing the resultant extract using HPLC. This method provides good sensitivity but requires lengthy liquid extraction steps and is not amenable to automation. To support the clinical trials and subsequent use of this and related compounds, a quantitation method is required which is reliable, straightforward, accurate and amenable to automation. ##STR1##